Microbial consortia for programmable output via photoautotroph-heterotroph interactions
Self-sustained, safe, stable and scalable microbial consortia (S 5 MicroCon) are described. The microbial consortia are regulated by photoautotroph-heterotroph interactions and RNA aptamer-based gene circuits. A rapid, high-throughput method for engineering RNA aptamer-based gene circuits (e.g. riboswitches) is also described.
1. An isolated microbial consortium, comprising a driver module, a process module and a control module, wherein:
the driver module comprises cyanobacteria comprising:
a first driver module nucleic acid construct that directs constitutive expression of a toxin encoded by ccdB, mazFa, relEs or vapC, and
a second driver module nucleic acid construct encoding a corresponding antitoxin encoded by ccdA, mazEa, relN or vapB, wherein expression of the antitoxin is regulated by a riboswitch responsive to p-amino-phenylalanine (pAF);
the process module comprises Escherichia coli or a species of Shewanella comprising:
a first process module nucleic acid construct encoding a reporter protein or an enzyme, wherein expression of the reporter protein or enzyme is regulated by a riboswitch responsive to theophylline (THP), and
a second process module nucleic acid construct encoding an acly homoserine lactone (AHL); and
the control module comprises a first control module nucleic acid construct encoding pAF, wherein expression of pAF is regulated by the AHL.
2. The microbial consortium of claim 1 , wherein the driver cyanobacteria comprises a species of Synechococcus, Cyanothece or Synechocystis.
3. The microbial consortium of claim 1 , wherein the driver module toxin and antitoxin are respectively encoded by ccdB and ccdA.
4. The microbial consortium of, claim 1 , wherein the process module comprises Escherichia coli.
5. The microbial consortium of claim 1 , wherein the reporter protein comprises a fluorescent protein.
6. The microbial consortium of claim 1 , wherein the process module further comprises a third process module nucleic acid construct that drives constitutive expression of a toxin encoded by ccdB, mazF, gef, yafO or parE, and a fourth process module nucleic acid construct encoding a corresponding antitoxin encoded by ccdA, mazE, sof, yafN or parD, respectively, wherein expression of the antitoxin is regulated by a riboswitch responsive to AHL.
7. The microbial consortium of claim 6 , wherein the process module toxin an antitoxin are respectively encoded by ccdB and ccdA.
8. The microbial consortium of claim 1 , wherein the first control module nucleic acid construct is located within the process module.
9. The microbial consortium of claim 1 , wherein the control module comprises Escherichia coli or a species of Shewanella comprising the first control module nucleic acid construct.
10. The microbial consortium of claim 9 , wherein the control module further comprises a second control module nucleic acid construct that drives constitutive expression of a toxin encoded by ccdB, mazF, gef, vafO or ParE, and a third control module nucleic acid construct ending a corresponding antitoxin encoded by ccdA, mazE, sof yafN or parD, respectively, wherein expression of the antitoxin is regulated by a riboswitch responsive to pAF.
11. The microbial consortium of claim 10 , wherein the control module toxin and antitoxin are respectively encoded by ccdB and ccdA.
12. A kit comprising the microbial consortium of claim 1 and growth media, a culture vessel, or both.
13. An isolated microbial consortium, comprising a driver module, a process module and a control module, wherein:
the driver module comprises a species of Synechococcus cyanobacteria comprising:
a first driver module nucleic acid construct that directs constitutive expression of a toxin encoded by ccdB and
a second driver module nucleic acid construct encoding a corresponding antitoxin encoded by ccdA, wherein expression of the antitoxin is regulated by a riboswitch responsive to p-amino-phenylalanine (pAF);
the process module comprises Escherichia coli comprising:
a first process module nucleic acid construct encoding a fluorescent protein, wherein expression of the fluorescent protein is regulated by a riboswitch responsive to theophylline (THP), and
a second process module nucleic acid construct encoding an acyl homoserine lactone (AHL); and
the control module comprises a first control module nucleic acid construct encoding pAF, wherein expression of pAF is regulated by the AHL.